The present invention relates to a novel filling composition for use in the column of a liquid chromatograph, and to a method for analytically examining a physiological specimen by liquid column chromatography.
It is extremely important for the diagnosis of diseases and the establishment of the guiding principle of treatments of the disease to obtain information on the morbid state by analysing the properties and the components of the patient's physiological specimens such as plasma, serum, cerebrospinal fluid, urine, etc.
Hitherto, such information has been obtained by various chemical and biochemical analytic means, however in the cases of elucidation of the morbid state of multifarious cases, a method of examination for obtaining exact information of the morbid state is required.
Particularly in cases of the diseases of the kidney and of the liver, according to the complicatedness of the morbid state, there has been a keen request for the development of new indications. That is, as the indications conventionally utilized of the morbid state, various determined values are mentioned, for example in the renal disease, the values representing the renal functions such as creatinine, uric acid and electrolytes in blood, protein and sugar in urine and pH of urine, and in the hepatic disease, the values representing the hepatic functions such as the activities of bioenzymes, for instance, GOT, GPT, LDH and LAP, and the components of bloods, for instance, protein and bilirubin.
These data of various determined values are playing important roles and are actually utilized for establishing the diagnosis and the guiding principles for treatment in their own ways.
However, recently it has been begun to point out that there is an intimate relationship between the uremic toxins and the morbid state of renal diseases, and the values determined by the conventional methods are not able to confirm the presence of the toxins, and moreover, no simple methods for detecting and confirming the toxins have yet been developed. Also in the hepatic diseases, in spite of the suggestion of the presence of various substances by abnormal metabolism and of the etiopathogenic substances, there are not suitable methods for detecting and confirming such substances at present.
In short, the present analytical methods of chemistry and biochemistry are not sufficient, although they are useful. And accordingly, the development of a suitable method for detecting and confirming the substance for supplying newer indications of the morbid state is keenly expected.
In consideration of the status quo, the utilization of the liquid chromatography which is one of analytical means based on the different principles from those of chemical and biochemical analytical methods has recently attracted the specialists' attention. Even thermally and chemically unstable substances are possibly determined by the liquid chromatography without being denaturation, and multiple components can be determined with a relatively small amount of the specimen, and accordingly, in principle, the applicability of the liquid chromatography in the fields of medicine and clinic is large enough. However, in order to put the liquid chromatography into practical use in the fields, there have been problems to be solved concerning the selection of the filling material, the establishment of conditions for separation in the chromatography and the pre-treatments adopted in accordance with the necessity.
For instance, Chang et al..sup.(1) tried to detect the toxins appearing in the blood of the patient suffering from the renal disease by using the liquid chromatography with columns filled with porous beads of cross-linked dextran. FNT (1) T. M. S. Chang et al. "Trans. Amer. Artif. Int. Organs", Vol. XX, page 364 (1974).
As a result, Chang et al. found that in the serum of the patient suffering from the renal disease different from the normal serum there are some substances which give specific peaks in the chromatogram obtained by their liquid chromatography, and suggested that the peaks include some harmful substances.
However, there are defects in the method of Chang et al., that is, the specific peaks obtained by the method are extremely broad and the method necessitates a relatively large amount of the serum specimen of 2 to 3 ml and moreover, it takes 4 to 7 hours in treating one specimen.
Accordingly, the method is not satisfactory as the clinical method of examination.
On the other hand, Furst.sup.(2) proposes a method of analysis of serum specimens by the high-speed liquid chromatography, however, although Furst's method was successful in shortening the time period for analysis, there is a defect of appearing many peaks over-lapping each other in the obtained chart of the chromatography to make the separation and identification of each component difficult. In addition, either of these methods has not been tried with an intension of applying to clinical examination. That is, although the analysis of bio-specimens by liquid chromatography has been tried, even if it is possible to detect some specific peaks in the chromatogram of a specimen from the patient suffering from a disease of the liver of the kidney, the result does not inform the relationship between the progress of the morbid state and the appearance of the peaks in the chromatogram. Accordingly, both methods of Chang and Furst have not been put into practical use. FNT (2) P. Furst, "Clinical Nephrology", Vol. 5(4), page 198 (1976)
We, in consideration of the status quo, have studied the methods for effectively examining clinically the physiological specimens from patients of, particularly, renal diseases, etc., and as a result, we have found that on the application of the liquid chromatography using a specified filling composition in its columns to such physiological specimen, it is possible to separate and detect the peaks in the chromatogram, which have relationship to the progress of the morbid state of the diseases within a short time period with only a small amount of the specimen.
Accordingly, the object of the present invention is to offer a novel filling composition for use in the column used for the liquid chromatography, particularly suitable for clinical examinations.
Another object of the present invention is to offer the chromatographical technique using the column utilizing the filling composition, particularly the technique of high-speed liquid chromatography. The other objects of the present invention will be made clear from the following description.